ABSTRACT: Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Currently, there is no effective therapy available, and the disease is universally fatal with a median overall survival of less than 15 months among patients who received standard treatments. Cancer immunotherapy holds great promises for GBM treatment, and growing evidence suggests that boosting the body's immune system can help eliminate highly aggressive and advanced tumors, including those resistant to conventional therapies. However, despite recent successes of cancer immunotherapies in other solid tumors, its effectiveness against GBM remains unclear. Furthermore, even for highly immunogenic tumors, only a small percentage of patients are likely responders. Therefore, there is an urgent need for the development of immunotherapies that are consistently effective for GBM patients. We have recently identified that the standard-of-care chemotherapeutic agent for GBM, temozolomide (TMZ), can induce immunogenic changes within the tumor via a mechanism that is distinctive and novel from their well-characterized DNA damaging effects. GBM cells exposed to TMZ experience a significant elevation in endoplasmic reticulum (ER) stress response with the corresponding translocation of ER chaperone protein, calreticulin (CRT) to the plasma membrane. CRT is a pro-phagocytic molecule that signals the recruitment for professional antigen presenting cells (APCs) for phagocytic clearance. However, TMZ-induced CRT translocation alone is insufficient to promote significant tumor clearance by APCs, suggesting that additional evasive signals are used by GBM to avoid eradication by the innate immune system. We subsequently showed that the anti-phagocytotic CD47 is overexpressed in GBM. Although the blockade of CD47 has been investigated as a potential therapy for multiple human cancers, its anti-tumor effect has been inconsistent. Therefore, based on these observations, we hypothesize that the simultaneous induction of CRT by TMZ and the blockade of CD47 signaling are both required to produce consistent and potent anti-GBM responses. Enhanced GBM phagocytosis and tumor-associated antigen cross-presentation by APCs subsequently heighten anti-tumor T cell adaptive response. Our proposal will mechanistically determine how TMZ with CD47 blockade primes the antigen-specific anti-GBM T cell responses, and evaluate the therapeutic utility of the combined treatment against TMZ-sensitive and TMZ-resistant GBMs. The proposed study will validate the clinical utility of the combined therapy in patient-derived GBM xenograft models implanted in mice with reconstituted human immune system. If successful, our study will demonstrate that conventional cytotoxic agents may possess immunogenic properties that can be harnessed to enhance GBM immunotherapy and generate relevant preclinical rationale to support further clinical investigations of TMZ and anti-CD47 combination for the treatment of GBM, particularly these resistant to TMZ.